Angiogenesis is a highly complex process of developing new blood vessels that involves the proliferation and migration of, and tissue infiltration by capillary endothelial cells from pre-existing blood vessels, cell assembly into tubular structures, joining of newly forming tubular assemblies to closed-circuit vascular systems, and maturation of newly formed capillary vessels.
Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing, as well as in pathological conditions such as tumor growth and in non-neoplastic diseases involving abnormal neovascularization, including neovascular glaucoma (Folkman, J. and Klagsbrun, M., Science, 235:442-7 (1987). Other disease states include but are not limited to, neoplastic diseases, including but not limited to solid tumors, atherosclerosis and other inflammatory diseases such as rheumatoid arthritis, and ophthalmological conditions such as diabetic retinopathy and age-related macular degeneration. Conditions or diseases to which persistent or uncontrolled angiogenesis contribute have been termed angiogenic dependent or angiogenic associated diseases.
One means for controlling such diseases and pathological conditions comprises restricting the blood supply to those cells involved in mediating or causing the disease or condition, for example, by occluding blood vessels supplying portions of organs in which tumors are present. Such approaches require the site of the tumor to be identified and are generally limited to treatment to a single site, or a small number of sites. An additional disadvantage of direct mechanical restriction of a blood supply is that collateral blood vessels develop, often quite rapidly, restoring the blood supply to the tumor.
Other approaches have focused on the modulation of factors that are involved in the regulation of angiogenesis. While usually quiescent, vascular endothelial proliferation is highly regulated, even during angiogenesis. VEGF is a factor that has been implicated as a regulator of angiogenesis in vivo (Klagsbrun, M. and D'Amore, P., Annual Rev. Physiol., 53: 217-39 (1991)).
An endothelial-cell specific mitogen, VEGF, acts as an angiogenesis inducer by specifically promoting the proliferation of endothelial cells. It is a homodimeric glycoprotein consisting of two 23 kD subunits. Four different monomeric isoforms of VEGF resulting from alternative splicing of mRNA have been identified. These include two membrane bound forms (VEGF206 and VEGF189) and two soluble forms (VEGF165 and VEGF121). VEGF165 is the most abundant isoform in all human tissues except placenta.
VEGF is expressed in embryonic tissues (Breier et al., Development, 114:521-32 (1992)), macrophages, and proliferating epidermal keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375-9 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev, 13:18-32 (1992)). In situ hybridization studies have demonstrated high levels of VEGF expression in a number of human tumor lines including glioblastoma multiforme, hemangioblastoma, other central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al., Nature, 359:845-8 (1992); Plate, K. et al., Cancer Res., 53:5822-7 (1993); Berkman, R. et al., J. Clin. Invest., 91:153-9 (1993); Nakamura, S. et al., AIDS Weekly, 13 (1) (1992)). High levels of VEGF expression has also been found in atherosclerotic lesions, plaques and in inflammatory cells.
VEGF mediates its biological effect through high affinity VEGF receptors which are selectively expressed on endothelial cells during, for example, embryogenesis (Millauer, B. et al. Cell, 72:835-46 (1993)) and tumor formation, and which have been implicated in modulating angiogenesis and tumor growth. These receptors comprise a tyrosine kinase cytosolic domain that initiates the signaling pathway involved in cell growth.
VEGF receptors typically are class III receptor-type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domains (Kaipainen et al., J. Exp. Med., 178:2077-88 (1993)). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Terman et al., Oncogene, 6:1677-83 (1991)). VEGF receptors include fins-like tyrosine kinase receptor (fit-1), or VEGFR-1, sequenced by Shibuya et al., Oncogene, 5:519-24 (1990), kinase insert domain-containing receptor/fetal liver kinase (KDR/flk-1), or VEGFR-2, described in WO 92/14248, filed Feb. 20, 1992, and Terman et al., Oncogene, 6:1677-83 (1991) and sequenced by Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026-30 (1991), although other receptors can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (fit-4), binds the VEGF homologues VEGF-C and VEGF-D and is important in the development of lymphatic vessels.
Release of VEGF by a tumor mass stimulates angiogenesis in adjacent endothelial cells. When VEGF is expressed by the tumor mass, endothelial cells adjacent to the VEGF+ tumor cells will up-regulate expression of VEGF receptors, e.g., VEGFR-1 and VEGFR-2. It is generally believed that KDR/VEGFR-2 is the main VEGF signal transducer that results in endothelial cell proliferation, migration, differentiation, tube formation, increase of vascular permeability, and maintenance of vascular integrity. VEGFR-1 possesses a much weaker kinase activity, and is unable to generate a mitogenic response when stimulated by VEGF, although it binds to VEGF with an affinity that is approximately 10-fold higher than KDR VEGFR-1 has also been implicated in VEGF and placenta growth factor (P1GF) induced migration of monocytes and macrophages and production of tissue factor.
High levels of VEGFR-2, for example, are expressed by endothelial cells that infiltrate gliomas (Plate, K. et al. (1992)), and are specifically upregulated by VEGF produced by human glioblastomas (Plate, K. et al. (1993)). The finding of high levels of VEGR-2 expression in glioblastoma associated endothelial cells (GAEC) suggests that receptor activity is induced during tumor formation, since VEGFR-2 transcripts are barely detectable in normal brain endothelial cells, indicating generation of a paracrine VEGF/VEGFR loop. This upregulation is confined to the vascular endothelial cells in close proximity to the tumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonal antibodies (mAbs) results in inhibition of the growth of human tumor xenografts in nude mice (Kim, K. et al. Nature, 362:841-4 (1993)), suggesting a direct role for VEGF in tumor-related angiogenesis.
Accordingly, VEGFR antagonists have been developed to treat vascularized tumors and other angiogenic diseases. These have included neutralizing antibodies that block signaling by VEGF receptors expressed on vascular endothelial cells to reduce tumor growth by blocking angiogenesis through an endothelial-dependent paracrine loop. See, e.g., U.S. Pat. No. 6,365,157 (Rockwell et al.), WO 00/44777 (Zhu et al.), WO 01/54723 (Kerbel); WO 01/74296 (Witte et al.), WO 01/90192 (Zhu), WO 03/002144 (Zhu), and WO 03/000183 (Carmeliet et al.).
VEGF receptors have also been found on some non-endothelial cells, such as tumor cells producing VEGF, wherein an endothelial-independent autocrine loop is generated to support tumor growth. For example, VEGF is almost invariably expressed by all established leukemic cell lines and freshly isolated human leukemias. Further, VEGFR-2 and VEGFR-1 are expressed by certain human leukemias. Fielder et al., Blood 89:1870-5 (1997); Bellamy et al., Cancer Res. 59728-33 (1999). It has been demonstrated that a VEGF/human VEGFR-2 autocrine loop mediates leukemic cell survival and migration in viio. Dias et al., J. Clin. Invest. 106:511-21 (2000); and WO01/74296 (Witte et al.). Similarly, VEGF production and VEGFR expression also have been reported for some solid tumor cell lines in vitro. (See, Sato, K. et al., Tohoku J. Exp. Med., 185: 173-84 (1998); Ishii, Y., Nippon Sanka Fujinka Gakkai Zasshi: 47: 133-40 (1995); and Ferrer, F. A. et al, Urology, 54:567-72 (1999)). It has further been demonstrated that VEGFR-1 Mabs inhibit an autocrine VEGFR/human VEGFR-1 loop in breast carcinoma cells. Wu, et al., “Monoclonal antibody against VEGFR1 inhibits flt1-positive DU4475 human breast tumor growth by a dual mechanism involving anti-angiogenic and tumor cell growth inhibitory activities,” AACR NCI EORTC International Conference on Molecular Targets and Cancer Therapeutics, Oct. 29-Nov. 2, 2001, Abstract #7.
There remains a need for agents0 which inhibit VEGF receptor activity to treat or prevent VEGF-receptor dependent diseases or conditions, by inhibiting, for example, pathogenic angiogenesis or tumor growth through inhibition of the paracrine and/or autocrine VEGF/VEGFR loop.